Rating scales are valuable tools in suicide research and can also be useful supplements to the clinical interview in suicide risk assessments. This study describes the psychometric properties of a Norwegian language version of the Suicide Assessment Scale Self-report version (SUAS-S).Participants were fifty-two patients  with major depression (53.8%), bipolar disorder (25.0%) and/or a personality disorder (63.5%) referred to a psychiatric outpatient clinic. The SUAS-S, the screening section of the Beck Scale for Suicidal Ideation (BSS-5), the Beck Depression Inventory (BDI), Beck’s Hopelessness Scale (BHS), the Symptom Check-List-90 R (SCL-90R) and the Clinical Global Impression for Severity of Suicidality (CGI-SS) were administered. One week later, the patients completed the SUAS-S a second time.Cronbach’s alpha for SUAS-S was 0.88 and the test–retest reliability was 0.95 (95% CI: 0.93– 0.97). SUAS-S was positively correlated with the BSS-5  for the study sample as a whole and for the suicidal (r = 0.52) and non-suicidal groups (r = 0.50) respectively. There was no difference between the SUAS-S and the BSS-5 in the ability to identify suicidality. This ability was more pronounced when the suicide risk was high. There was a substantial intercorrelation between the score on the SUAS-S and the BDI (0.81) and the BHS (0.76). The sensitivity and specificity of the SUAS-S was explored and an appropriate clinical cut-off value was assessed.The study revealed good internal consistency, test–retest reliability and concurrent validity for the Suicide Assessment Scale Self-report version. The discriminatory ability for suicidality was comparable to that of the BSS-5. Assessment of suicide risk is one of the most challenging tasks that clinicians working in mental health care face. The difficulty of the task derives from problems with the definition of suicidality , the facThe SUAS-S is a 20-item self-report rating scale measuring the patient’s attitude towards suicide, suicide-related behaviour and suicidal ideation on the day of reporting and during the previous seven days. Each item is scored in the range of 0–4 on a Likert-type scale and resulting in a scale sum score with a range of 0–80. The scale is designed to measure levels of suicidality and to be sensitive to temporal changes in suicide-related symptoms . The SUAFifty-five patients consecutively referred to a psychiatric outpatient clinic from January 2008 until January 2009, were asked to participate in the project. Fifty-three (96%) of the patients provided informed consent and were included in the study. Of these, one patient dropped out before all scales were completed and was not included in the further analysis. Patients with an on-going substance abuse and/or psychotic disorder were not eligible to participate in the study. Patients were recruited from a non-emergency setting.Deliberate self-harm (DSH) was defined as “an act with non-fatal outcome in which an individual deliberately initiated behaviour with the intention to cause self-harm, for example, self-cutting or jumping from a height, by ingestion of an illicit drug, a non-ingestible substance or an excess of a prescribed substance”,11. Suict-test or Mann–Whitney U test. Internal consistency was computed as the Cronbach’s alpha coefficient, and test–retest reliability was measured with Intra Class Correlation (ICC) with 95% Confidence Intervals (CIs). Associations between the sum scores on the SUAS-S and BSS-5, and their precision, were estimated by Pearson’s or Spearman’s correlation coefficient and bootstrapped 95% CI with 10,000 replications. Discriminatory ability of suicidality for the SUAS-S and BSS-5 sum scores was examined and compared through the use of nonparametric receiver operating characteristic (ROC) curve analyses. The tests were two-sided, and the significance level was set at 0.05. The analyses were performed with STATA 11 [Means and standard deviations, or medians and quartiles, were given for sum scores. The chi-squared test was used to assess differences in distribution between the groups for categorical variables. Differences in sum scores of two independent groups were tested by two-sample STATA 11  and the STATA 11 .The study was approved by the Regional Committee for Medical Research Ethics, South-East Norway. All patients gave written informed consent.The sample consisted of 33 (63.5%) females and 19 males (36.5%) with a mean age of 39.3 years . Thirty-four patients (65.4%) had been in psychiatric treatment in total for more than five years, and 11 (21.2%) for less than a year. The mean GAF score was 48.6 (SD = 4.6); there were no gender differences. The mean SCL-90-R score was 1.5 (SD = 0.7), with no significant gender differences. Twenty-two (42.3%) of the patients, 16 females and six males, reported a history of DSH. In 15 (68.2%) of these cases, the criteria for a SA were met. Five patients reported having had a DSH episode during the last six months. The median number of DSH episodes per patient was one, with a range of 0–30.All but one patient fulfilled the criteria for at least one axis I disorder. Major Depressive Disorder (MDD) was diagnosed in 28 patients (53.8%), Bipolar Disorder type II in 13 cases (25.0%) and Dysthymia in 4 cases (7.5%). These diagnostic groups were merged into one group, “Affective Disorders” (AD) (n = 45). The residual group (n = 7) was labelled “Other Axis I Disorders”. Thirty-three patients (63.5%) had an axis II diagnosis, of whom five (9.6%) had a Cluster A disorder, 11 (21.2%) a Cluster B disorder, 15 (28.8%) a Cluster C disorder, and two patients had Personality Disorder Not Otherwise Specified (PF-NOS).The mean SUAS-S score was 31.0 (SD = 9.3) in females and 28.1 (SD = 12.7) in males. No significant correlation was found between age and the SUAS-S score. Patients with AD had a significantly higher median SUAS-S score  than patients with other axis I disorders , (p = 0.018).Internal consistency, as measured by Cronbach’s alpha coefficient, was found to be 0.88 (95% CI: 0.82–0.92). The test–retest reliability expressed by the ICC was 0.95 (95% CI: 0.93–0.97). Concurrent validity was examined by pairwise correlations with 95% CIs between SUAS-S, BSS-5, BDI and BHS-scores for all patients, as shown in Table SUAS-S was generally strongly correlated with these measures, but somewhat weaker with BSS-5. The non-linear co-variation between the BSS-5 and SUAS-S scores is more clearly shown in Figure The sensitivity and specificity of the SUAS-S was explored as tabulated in Table A cut-off value of 29 on the SUAS-S scale yielded a sensitivity of 80% and specificity of 56%. The negative and positive predictive probabilities of this chosen cut-off point were 75% and 63%, respectively.Descriptive and association analyses of the five areas of the SUAS-S scale, with a categorical classification as suicidal on the CGI-SS scale as the dependent variable, showed that the sum scores of the area “suicidal thoughts and behaviour” (SUAS-S items 16–20) was strongly associated with this variable (p < 0.001). The other three areas, “bodily states”, “control and coping” and “emotional reactivity” were not significantly associated with this dependent variable. The results of analyses are shown in Table Pairwise associations between the BSS-5, depression, hopelessness and the sum scores of the five areas are shown in Table Correlation analyses showed that the SUAS-S areas of “affect” and “suicidal thoughts and behaviour” and “emotional reactivity” were strongly and significantly associated with depression and hopelessness . The “bodily states” area was significantly correlated with depression (expressed by the BDI sum score) but not with hopelessness (expressed by the BHS sum score). The association was strongest between the sum scores of the area “suicidal thoughts and behaviour” and BSS-5 . The results of correlation analyses are shown in Table Figure The psychometric properties of the Norwegian language version of the SUAS-S in a non-emergency psychiatric outpatient setting were found to be good, with internal consistency and test–retest reliability properties comparable to the original version. Likewise, the construct validity was found to be favourable. Of the five areas in the SUAS-S, the “suicidal thoughts and behaviour” subscale was significantly and strongly associated with the clinician’s global assessment of suicidality. The findings of a strong correlation between the SUAS-S and BSS-5 (0.66), and no significant difference between the ROCs for these two scales Figure , suggestThe limited number of patients excluded the possibility to study the SUAS-S properties in more narrow diagnostic subgroups. The selection criteria applied in this study with exclusion of on-going substance abuse and psychotic disorders limits the external validity of findings to non-psychotic and non-abusing patients in non-emergency psychiatric evaluation settings.The Norwegian language version of the SUAS-S was found to possess psychometric properties equal to the original version and seems to be a valid and reliable instrument in the assessment of suicide risk and depression among patients in the mental health care system. The SUAS-S also has the advantage of open access with no copyright costs.All authors declare that they have no conflicts of interest.BOK and LM designed the study. BOK collected the data and LMD performed the statistical analyses and contributed to the writing of the manuscript. BOK, LM, LSM and FAW wrote the manuscript and all authors approved the final version.. F. A. Walby MA is a researcher at the National Centre for Suicide Research and Prevention, Institute of Clinical Medicine, University of Oslo and chief psychologist in the Department of Psychiatry, Diakonhjemmet Hospital, Oslo. L.M. Diep MSc is a statistician at Oslo University Hospital, the National Centre for Suicidal Research and Prevention at University of Oslo, and the Institute for Community Medicine at University of Oslo, Norway.B.O.Koldsland MD is a senior psychiatrist at Vestre Viken Health Trust, Ringerike Hospital psychiatric outpatient clinic, and affiliated with the National Centre for Suicide Research and Prevention, University of Oslo. L. Mehlum MD PhD is a professor and head of the National Centre for Suicide Research and Prevention, Institute of Clinical Medicine, University of Oslo. L.S. Mellesdal RN MSc is a research fellow in the Psychiatric Division of Haukeland University Hospital, BergenAppendix. SUAS.Click here for file


In non-gastrointestinal stromal tumor soft tissue sarcoma (non-GIST STS) optimal treatment is surgery with wide resection margins. Vascular endothelial growth factors (VEGFs) and receptors (VEGFRs) are known to be key players in the initiation of angiogenesis and lymphangiogenesis. This study investigates the prognostic impact of VEGFs and VEGFRs in non-GIST STS with wide and non-wide resection margins.Tumor samples from 249 patients with non-GIST STS were obtained and tissue microarrays were constructed for each specimen. Immunohistochemistry was used to evaluate the expressions of VEGF-A, -C and -D and VEGFR-1, -2 and -3.In the univariate analyses, VEGF-A (P = 0.040) in the total material, and VEGF-A (P = 0.018), VEGF-C (P = 0.025) and VEGFR-3 (P = 0.027) in the subgroup with wide resection margins, were significant negative prognostic indicators of disease-specific survival (DSS). In the multivariate analysis, high expression of VEGFR-3  was an independent significant negative prognostic marker for DSS among patients with wide resection margins.VEGFR-3 is a strong and independent negative prognostic marker for non-GIST STSs with wide resection margins. Soft tissue sarcomas (STS) originate from the mesenchymal lineage, and thus share a similar ancestry Classically STSs have been treated as a single group. This is mainly because the low incidence makes it difficult to conduct decently powered studies on the individual histological entities. With the emerging knowledge of cellular processes and the increasing knowledge about common and uncommon genetic translocations the last decade, it is now clear that the picture might be more intricate. For instance, Ewing family tumors, synovial sarcoma, rhabdomyosarcoma, dermatofibrosarcoma protuberens and others have distinct genetic translocations The main treatment of sarcomas is surgical resection, and wide resection margins are considered one of the most important prognostic factors Angiogenesis inhibitors provide a new and exciting therapeutic option for patients with STS High levels of VEGF-A in tumors and blood samples from STS patients have previously been associated with higher tumor grade, increased tendency to metastasis, reduced response to treatment, lower overall survival (OS) and increased risk of recurrence In this study, the aim was to assess the prognostic impact of VEGF-A, -C, -D and VEGFR-1. -2 and -3 in non-GIST STS patients with wide and non-wide resection margins.Primary tumor tissue from anonymized patients diagnosed with non-GIST STS at the University Hospital of North-Norway and the Hospitals of Arkhangelsk county, Russia, from 1973 through 2006, were collected. In total 496 patients were registered from the hospital databases. Of these 247, patients were excluded from the study due to: missing clinical data (n = 86) or inadequate paraffin-embedded fixed tissue blocks (n = 161). Thus 249 patients with complete medical records and adequate paraffin-embedded tissue blocks were eligible.This report includes follow-up data as of September 2009. The median follow-up was 37.6 (range 0.1–391.7) months. Complete demographic and clinical data were collected retrospectively. Formalin-fixed and paraffin-embedded tumor specimens were obtained from the archives of the Departments of Pathology at the University Hospital of North-Norway and the Hospitals of Arkhangelsk County. The tumors were graded according to the French Fédération Nationale des centres de Lutte Contre le Cancer (FNCLCC) system and histologically sub typed according to the World Health Organization guidelines All sarcomas were histologically reviewed by two trained pathologists  and the most representative areas of tumor cells  were carefully selected and marked on the hematoxylin and eosin (H/E) slide and sampled for the tissue microarray (TMA) blocks. The TMAs were assembled using a tissue-arraying instrument . The Detailed methodology has been previously reported To include all core samples, 12 TMA blocks were constructed. Multiple 5-µm sections were cut with a Micron microtome (HM355S) and stained by specific antibodies for immunohistochemistry (IHC) analysis.The applied antibodies were subjected to in-house validation by the manufacturer for immunohistochemical analysis on paraffin-embedded material. The antibodies used in the study were as follows: VEGF-A , VEGF-C , VEGF-D , VEGFR-1 , VEGFR-2 , and VEGFR-3 .Sections were deparaffinized with xylene and rehydrated with ethanol. Antigen retrieval was done by placing the specimen in 0.01 mol/L of citrate buffer at pH 6.0 and exposed to repeated (twice) microwave heating of 10 min  at 450 W. VEGF-D was heated for 45 min in a water bath in 0.01 mol/L of citrate buffer. The DAKO EnVision+ System-HRP kit (diaminobenzidine) was used for endogen peroxidase blocking. As negative staining controls, the primary antibodies were replaced with the primary antibody diluents. Primary antibodies were incubated for 30 min in room temperature . The DAKO EnVision+ System-HRP kit (diaminobenzidine) was used to visualize the antigens. This was followed by the application of liquid diaminobenzidine and substrate-chromogen, yielding a brown reaction product at the site of the target antigen. Finally, all slides were counterstained with hematoxylin to visualize the nuclei. For each antibody, included negative staining controls, all TMA staining were done in a single experiment.The ARIOL imaging system  was used to scan the slides of antibody staining of the TMAs. The slides were loaded in the automated slide loader (Applied Imaging SL 50) and the specimens were scanned at low resolution (1.25×) and high resolution (20×) using the Olympus BX 61 microscope with an automated platform (Prior). Representative and viable tissue sections were scored manually on computer screen semi quantitatively for cytoplasmic staining. The dominant staining intensity was scored as: 0 =  negative; 1 =  weak; 2 =  intermediate; 3 =  strong. All samples were anonymized and independently scored by two trained pathologists . When assessing a variable for a given core, the observers were blinded to the scores of the other variables and to outcome. Mean score for duplicate cores from each individual was calculated separately.High expression in tumor cells was defined as score ≥1.5  and ≥1 (VEGF-C) .All statistical analyses were done using the statistical package SPSS , version 16. The IHC scores from each observer were compared for interobserver reliability by use of a two-way random effect model with absolute agreement definition. The intraclass correlation coefficient (reliability coefficient) was obtained from these results. The Chi-square test and Fishers Exact test were used to examine the association between molecular marker expression and various clinicopathological parameters. Univariate analyses were done using the Kaplan-Meier method, and statistical significance between survival curves was assessed by the log rank test. DSS was determined from the date of diagnosis to the time of cancer related death. To assess the independent value of different pretreatment variables on survival, in the presence of other variables, multivariate analyses were carried out using the Cox proportional hazards model. Only variables of significant value from the univariate analyses were entered into the Cox regression analyses. Probability for stepwise entry and removal was set at .05 and .10, respectively. The significance level used for all statistical tests was P<0.05.The National Data Inspection Board and The Regional (Northern Norway) Committee for Research Ethics approved the study.The clinopathological variables are summarized in Interobserver scoring agreement was tested for one ligand (VEGF-C) and one receptor (VEGFR-3). The intraclass correlation coefficients were 0.810 for VEGF-C (P<0.001) and 0.834 for VEGFR-3 (P<0.001) indicating good reproducibility between the two investigating pathologists.VEGF/VEGFR expression was observed in the cytoplasm of tumor cells. For the ligand and receptor expressions we found the following correlation with malignancy grade: High VEGF-A expression, grade 1: 29%, grade 2: 48%, grade 3: 56% (P = 0.005); High VEGF-C expression, grade 1: 24%, grade 2: 41%, grade 3: 45% (P = 0.032); High VEGFR-1 expression, grade 1: 27%, grade 2: 36%, grade 3: 48% (P = 0.034); High VEGFR-2 expression, grade 1: 12%, grade 2: 27%, grade 3: 39% (P = 0.001).The influence on DSS by the VEGFs and VEGFRs are given in Results of the multivariate analyses are presented in In this study we observed that high expression of VEGFR-3 was a significant independent negative prognostic indicator of DSS in non-GIST STS patients with wide resection margins. Although there have been prior evaluations of the VEGF axis in STSs, these have primarily been focused on VEGF-A. Herein, we have presented a large-scale study of the prognostic impact of VEGF-A, -C and -D and VEGFR-1-3 in non-GIST STS patients. To our knowledge, this is the first evaluation of these pathways according to resection margins.The major weakness of this study, normally seen in sarcoma studies in general, is the heterogeneity of the sarcoma population. Even with a relatively large sample cohort with regard to non-GIST STSs, the numbers are too small to do meaningful explorations according to histological subgroups, at least with respect to multivariate analysis.Wide resection margins have been demonstrated to give the best overall survival, with more modest results for marginal and particularly intralesional resections 3 in size This is the first report of VEGFR-3 expression being an independent negative prognostic marker in non-GIST with wide resection margins. VEGFR-3 is a tyrosine-kinase receptor that is activated by VEGF-C and -D. The VEGFR-3/VEGF-C/-D system is considered the main pathway responsible for developing lymphatic vessels In the presented population with wide resection margins, tumor VEGF-C expression was a significant negative prognostic marker for DSS. To our knowledge, only one small study has previously reported on this relationship in STSs. In 45 patients with undifferentiated pleomorphic sarcoma  and neurogenic sarcoma, Hoffmann et al. concluded surprisingly that high expression of VEGF-C mRNA led to a longer overall survival We found VEGF-A expression in tumor tissue to be a significant negative prognostic marker for DSS in univariate analyses, both in the total material and in the subgroup with wide resection margins. Further, we found that VEGF-A and its corresponding receptors VEGFR-1 and -2, showed significant correlations with histological tumor grade, in accordance with previously published studies Antibodies targeting the VEGF/VEGFR systems are readily available, and clinical trials with such agents have been initiated in several cancer types As our data are prognostic and not mechanistic we cannot conclude on which pathways are operative in non-GIST STSs expressing VEGFs and VEGFRs. Nevertheless, it can be deduced that VEGFs and VEGFRs play critical roles in sarcoma progression and prognosis. But whether angiogenic ligands and receptors may have predictive effects with respect to therapy remains unanswered. The mechanistic impacts of angiogenesis, lymphangiogenesis, autocrine versus paracrine pathways as well as the relevance of constitutively activated receptors have to be further clarified. Consequently, future translational research in this field is needed.


The 16/6-idiotype (16/6-Id) of the human anti-DNA antibody was found to induce experimental lupus in naïve mice, manifested by production of autoantibodies, leukopenia and elevated inflammatory markers, as well as kidney and brain involvement. We assessed behavior and brain pathology of naive mice injected intra-cerebra-ventricularly (ICV) with the 16/6-Id antibody.C3H female mice were injected ICV to the right hemisphere with the human 16/6-Id antibody or commercial human IgG antibodies (control). The mice were tested for depression by the forced swimming test (FST), locomotor and explorative activity by the staircase test, and cognitive functions were examined by the novel object recognition and Y-maze tests. Brain slices were stained for inflammatory processes.P = 0.012). Similarly, the preference for spatial novelty in the Y-maze test was significantly higher in the control group compared to the 16/6-Id-injected mice . Depression–like behavior and locomotor activity were not significantly different between the16/6-Id-injected and the control mice. Immunohistochemistry analysis revealed an increase in astrocytes and microglial activation in the hippocampus and amygdala, in the 16/6-Id injected group compared to the control.16/6-Id injected mice were cognitively impaired as shown by significant differences in the preference for a new object in the novel object recognition test compared to controls (Passive transfer of 16/6-Id antibodies directly into mice brain resulted in cognitive impairments and histological evidence for brain inflammation. These findings shed additional light on the diverse mosaic pathophysiology of neuropsychiatric lupus.http://www.biomedcentral.com/1741-7015/11/91See related Commentary article:  Neuropsychiatric systemic lupus erythematosus (NPSLE) refers to a complex set of syndromes involving the central nervous system (CNS) in up to 56% of lupus patients-5. Due tKlebsiela polysaccharides and Mycobacterium tuberculosis glycoproteins, brain glycolipids and tumor cells[ex vivo. The presence of circulating 16/6-Id was detected in patients with other autoimmuine diseases as well [More than 20 brain specific and non-specific autoantibodies have been proposed to be involved in the mechanism of NPLSE, includior cells. The preor cells,24. Elevor cells. Depositor cells,26,27, alerosis),29. Immulerosis)-32. In aad libitum access to food and water. The Sheba Medical Center Animal Welfare Committee approved all procedures.Three-month-old, female C3H mice were obtained from Harlan Laboratories, Jerusalem, Israel, and were housed in the animal facility at Sheba Medical Center. The mice were raised under standard conditions, 23 ± 1°C, 12-hour light cycle (6:30 am to 6:30 pm) with The human monoclonal anti–DNA antibodies were produced by a hybridoma derived from fusion of the GM4672 lymphoblastoid cell line and peripheral blood or splenic lymphocytes obtained from three lupus patients. The human mAb that bears the 16/6-Id (IgG1/k) has been characterized previously. The mAbet al.[The injection process is based on a detailed protocol reported by Shoenfeld et al.. Mice weTwenty-one CH3 mice were injected ICV to the right hemisphere, 11 with human 16/6-Id antibodies and 10 with commercial human IgG antibodies (control). The forced swimming test (FST) was performed at Days 16 and 23 after antibody injection, the staircase test at Days 14 and 26, the novel object recognition at Days 19 and 20 and the Y-maze test at Day 21. At Day 24, under anesthesia, a systemic perfusion was performed, and the brains were collected. Immunofluorescence staining was performed to detect markers of inflammation or neuronal degeneration (see below).et al.‘s description[This test is based on Porsolt cription. Mice weet al.[Locomotor and explorative activity was evaluated by the staircase test, as described previously by Katzav et al.. This teet al.[This is a visual recognition memory test based on a method described by Tordera et al.. The appThe Y maze test was used to assess spatial memory. It was comprised of three arms, built of black Perspex. Each arm was 8 × 30 × 15 cm at an angle of 120° from the others. One arm was randomly selected as the start arm. Each mouse was placed twice in the start arm. On the first trial, lasting for five minutes, one of the other two arms was randomly chosen to be blocked whereas on the second trial, lasting for two minutes, both arms were open. The two trials were separated by a two-minute interval, during which the mouse was returned to its home cage. The time spent in each of the arms was measured. Between each trial and between each mouse, the maze was cleaned with a 70% alcohol solution and dried. Discrimination of spatial novelty was assessed by a preference index: time inThe mice were anesthetized by an i.p. injection of ketamine (100 mg/kg) and xylazine (20 mg/kg) and sacrificed by transcardiac perfusion with phosphate buffered saline (PBS) followed by perfusion with 4% paraformaldehyde  in phosphate buffer . After perfusion, the brain was quickly removed and fixed overnight in 4% PFA  at 4°C. On the following day, the brain was cryoprotected by immersion in 30% sucrose in 0.1M PO4 (pH 7.4) for 24 to 48 hours at 4°C before brain cutting.Frozen coronal sections (30 to 50 μm) were cut on a sliding microtome , collected serially and kept in a cryoprotectant at −20°C until staining. Staining was performed as follows. Six mice (three IgG control and three 16/6 Id) were used for immunohistochemistry. Brain sections were stained free-floating, incubated with the first antibodies overnight at 4°C. The slices were then washed in PBS + 0.1% Triton X-100, and incubated at room temperature for one hour with the corresponding fluorescent chromogens-conjugated secondary antibody. Sections were stained for specific antigens with antibodies against activated microglia  and astrocytes . Counter staining was performed with Hoechst .T-test. Significant results were determined as P <0.05.Results are expressed as the mean ± SEM. The differences in mean for average immobility time in the FST, the staircase test parameters (number of rearing and stair-climbing events), novel object recognition and Y-maze tests were evaluated by P = 0.012), while no difference in the preference was seen in the mice injected with 16/6-Id . This suggests a specific visual recognition memory impairment in the 16/6-Id mice. Similarly, cognitive performance in the Y-maze test is presented as a preference index for new  in both groups .The results of cognitive performance in the novel object recognition test are presented as the proportion time spent near objects (new and old) in both groups Figure . There wP = 0.159 by t-test) and 182.5 ± 45.4 vs. 205.7 ± 42.7 sec (P = 0.238 by t-test) on Days 16 and 24, respectively.In the forced swimming test there was no significant difference between 16/6-Id injected and control mice in depression-like behavior at Days 16 and 24 after injection. Average immobility times of the control mice vs. 16/6-Id injected mice were 117.6 ± 65.9 vs. 160 ± 72.8 (P >0.016). The results also did not change from Day 14 to 26.In the staircase test, there was no significant difference between the average rearing and stair-climbing counts, among mice from control-IgG vs. 16/6-Id . The 16/6-Id injected mice demonstrated increased microglial activation (Iba-1 staining), at the hippocampus  as well as the amygdala, compared to IgG control Figure .In the present study we have observed that passive transfer of 16/6-Id antibodies directly to mice brains resulted in a selective cognitive impairment, expressed as visual recognition and spatial memory deficits. Depressive behavior (FST) and locomotor activity (staircase test) were not altered in the 16/6-Id injected mice, when compared to the control group. Our findings suggest that 16/6-Id antibodies may have a role in the pathogenesis of cognitive impairment observed in some patients with SLE.et al. demonstrated that anti-ATP synthase autoantibodies, purified from Alzheimer’s disease patients, can lead to cognitive impairment and hippocampal neuron apoptosis in naïve mice[Immunostaining of brain sections from both groups revealed increased presence of activated microglia and astrocytes, in the hippocampal region of the 16/6-Id injected mice, compared to the controls. The hippocampus has an important function in memory processing, therefore, its damage by an inflammatory processes may affect cognitive performance in the 16/6-Id injected mice. Astrocytes in steady state conditions are mainly responsible for biochemical support and several other chemical roles such as maintenance of extracellular ion balance. However, in special situations, astrocytes may increase in number as an inflammatory reaction aimed for scaring and repairing CNS tissue. Microglia serve as scavengers and are activated in an inflammatory reaction. The presence of more astrocytes (gliosis) or the activation of microglia in brain tissue can implicate an inflammatory state. Our hypothesis regarding the pathogenesis of 16/6-Id antibodies induced-brain impairment includes several mechanisms: 1) Neuronal degeneration may be caused by direct or indirect injury to hippocampal area. For example, recently Berry ïve mice. Other nïve mice,40. 2) Nïve mice. 3) Braiet al., anti-DNA antibodies which can cross-react with the NR2 - anti-NMDA receptor were found in the sera, CSF and brains of SLE patients[in vivo and in vitro[et al. added another layer to the current understandings regarding the role of different auto-antibodies in the pathogenesis of NPSLE. Another technique to bypass the BBB was used by us in several experiments. In the ICV technique, antibodies were injected directly into the lateral ventricle in the mouse brain, allowing antibody dispersal throughout the brain tissue. In our previous studies, an experimental NPSLE was induced by passive transfer of anti-ribosomal-P antibodies directly to mice brains[We have extensively studied the pathogenesis of different autoantibodies and their influence on the brain. Injection of anti-ribosomal-P antibodies ICV to naïve mice resulted in depressive-like behavior in these mice,43. In apatients,47. Thes in vitro,47. In t in vitro, while oe brains, as welle brains.via the idiotypic network, in which injection of human anti-DNA 16/6-Id mAbs induces the generation of anti-Id, and anti-anti-Id, and so on. The production of 16/6-Id was found to be induced also by several infectious agents ; this cerculosis.The finding, that 16/6-Id antibodies were detected in other autoimmune diseases, such as PM/DM and scleroderma without them expressing central nervous symptoms is interesting. Perhaps, in some diseases  a variety of systemic factors enable the altering of BBB permeability. These factors may include other circulating antibodies, inflammatory elements, as well as vasogenic agents, growth factors and free radicals. This phenomenon is not unusual in the autoimmunity field, for instance, anti-Ro antibodies are associated with myositis or sub-acute skin manifestations in some SLE patients and not in Sjogren patients.The current finding, that the 16/6-Id is related to spatial novelty and visual recognition memory impairments in mice, may attest for immune-mediated damage to brain areas relevant for these functions. There is a wide agreement that spatial long-term memory and object recognition is dependent on the functioning of the hippocampal region. Taken tPassive transfer of anti-DNA 16/6-idiotype directly to mice brains resulted in cognitive impairment, supported by cognitive testing impairments, and changes in brain histological analysis. Therefore, the 16/6-idiotype may have a role in cognitive decline, as well as other neuropsychiatric manifestations, which are found in lupus patients.anti-ssDNA: Anti-single-stranded-DNA; BBB: Blood–brain barrier; CNS: Central nervous system; FST: Forced swimming test; ICV: Intra-cerebra-ventricularly; i.p: Intra-peritoneal; LPS: Lipopolysaccharide; mAb: Monoclonal antibody; MRI: Magnetic resonance imaging; NMDA: N-methyl-D-aspartate; NPSLE: Neuropsychiatric systemic lupus erythematosus; PBS: Phosphate buffered saline; PFA: Paraformaldehyde; SLE: Systemic lupus erythematosus; 16/6-Id: 16/6-idiotypeThe authors declare that they have no competing interests.SK participated in the immunohistochemical and behavioral studies and drafted the manuscript. AK participated in the immunohistochemical and behavioral studies and helped to draft the manuscript. MTA participated in the immunohistochemical studies and performed the statistical analysis. MLR and YZ participated in the behavioral studies. NAL participated in the behavioral studies and helped to draft the manuscript. MB participated in the design and helped to draft the manuscript. JMA participated in the design and coordination of the study. EM and JC participated in the design and coordination of the study and helped to draft the manuscript. YS conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/90/prepub


There are a limited number of studies correlating the different stages of dendritic cells (DC) maturation with cytokines in individuals presented chronic periodontitis (CP). The aim of the study was to evaluate the correlation among the expression of IL-2, IL-10, IL-4, IL-6, IFN- Chronic periodontitis (CP) Myeloid DCs, also known as conventional DCs, present a strong capability of capturing antigens, which enables them to stimulate T cells CD4 T-cells can be subdivided into Th1, Th2, Th17, and Treg subsets on the basis of their pattern of cytokine production The role of T-helper cells and DCs in the pathogenesis of CP has not yet been fully clarified. There are a limited number of studies correlating the different stages of DC maturation with pro-inflammatory (IL-6 and tumor necrosis factor (TNF)-α), Th1 (IL-2 and interferon (IFN)-This study was approved by the Committee of Ethics in Research from Universidade Federal de Minas Gerais (UFMG), Brazil (423/11). Thirty patients provided written informed consent to participate in this study. All individuals received a full-mouth periodontal examination in which probing depth (PD), clinical attachment level (CAL), bleeding on probing, and oral hygiene index were determined Six samples were collected from the mucosa overlapping the third molar that had been recommended for extraction. These samples were used to control the cytokine levels.Multiple gingival tissue cytokines  using the Image Tool software . The mean number of cells per unit area (cell number per square millimeters) was obtained. Inflammatory infiltrate density was correlated with periodontal indexes, densities of DCs, and cytokine levels. Furthermore, data were dichotomized by medians: mild and intense inflammatory infiltrate The inflammatory infiltrate in gingival tissue was measured using hematoxylin and eosin stained sections . These sAn immunohistochemical reaction was performed using the streptavidin-biotin standard protocol. Anti-CD1a and anti-Factor XIIIa are considered markers for immature DCs. Anti-CD1a is typical epidermal Langerhans cells (LC) characterized by the expression of Birbeck granules, while Factor XIlla is described as dermal dendritic cells Densities of immunolabeled cells (cell number per square millimeters) were calculated for CD1a, Factor XIIIa, and CD83. Positive cell counts were restricted to immunolabeled cells that exhibited well-defined nuclei. The slices were digitized with a microscope  at 400x magnification and interfaced to a computer. Cell counts were performed throughout the sections by a blinded examiner (GRS). Areas were delineated using a mouse and measured using the software AxioVision .SPSS statistic software  was used for statistical analyses. Normal distribution was tested using the Shapiro-Wilks procedure. In samples with a normal distribution, the Student t test was applied and P values were <0.05. Kruskal–Wallis and Mann-Whitney U tests were used to analyze the samples with abnormal distributions. After Bonferroni correction, the statistical significance was achieved when P values were <0.016. For correlation tests, all samples demonstrated abnormal distribution, and Spearman correlation was applied; the α level was set to 0.05. The correlation was graded according to the Cohen classification as weak (<0.30), moderate (0.30 to 0.50), or strong (>0.50) Sample calculation considered both type I and II errors. For this, we assumed a 95% confidence interval, 80% power of test, and parameters of values of levels of IL17A obtained in study of periodontal disease of Behfarnia et al. The studied population presented a mean age of 44.50±12.19 years . Periodontal clinical parameters of the samples are presented in All regions of the gingival tissue were evaluated for each antibody. Factor XIIIa+ immature DCs could be observed only in the LP ; CD1a+ iRegarding possible altered phenotypes between the female and male genders, when comparing the density of inflammatory infiltrate cells and the density of immature and mature DCs, no significant differences between genders could be observed.The present study evaluated whether or not the density of inflammatory infiltrate cells influenced the quantity of DCs. Eleven samples were grouped in mild inflammatory infiltrate and eleven in intense inflammatory infiltrate. The density of Factor XIIIa+ immature DCs was higher in individuals presenting an intense, as compared to mild, inflammatory infiltrate density (P<0.05) . For thiPro-inflammatory (IL-6 and TNF-α), Th1 (IL-2 and INF- (n = 6) . IL-2, Ied to NM . In addied to NM . No diffPositive correlation (P<0.05) could be observed between IL-6 and the density of CD1a+ immature DCs, as well as the density of inflammatory infiltrate cells, in both the SE and LP. A positive correlation (P<0.05) could also be observed between IL-6 and the percentage of sites with PD>4. By contrast, a negative correlation (P<0.05) could be identified between IL-2 and the percentage of sites with CAL>3.Positive correlations (p<0.05) could be observed between the percentage of sites with PD>4 and the density of inflammatory infiltrate cells, CD1a+ immature DCs in the SE, and Factor XIIIa+ immature DCs. Moreover, a positive correlation (p<0.05) could also be identified between the percentage of sites with CAL>3 and the density of inflammatory infiltrate cells. However, negative correlations (p<0.05) could be observed between the number of teeth with CAL>3 and CD1a+ immature DCs in the SE, as well as between the number of teeth with CAL>5 and CD1a+ immature DCs in the SE and LP.in vitro studies have shown that IL-6 plays a major role in maintaining immature DCs and in blocking DC maturation. IL-6-mediated negative feedback may well contribute to the down-regulation of the immune response initiated by pathogens or in persistent infections DCs are the most potent antigen-presenting cells (APCs), whose function is to initiate an adaptive immune response. Activation and maturation of DCs are induced by infectious agents and inflammatory products In addition, it is known that macrophages are important APCs. It was demonstrate that these cells can contribute to innate immunity employing various cytokines and chemokines IL-6 levels showed no increase when compared with NM, whereas the levels of IL-2, TNF-α, INF-In line with these results, the IL-6 expression proved to be positively correlated with PD. Guillot et al. Th1-related cytokines are related to the early process of periodontal disease Differences in cytokines levels can be observed in accordance with the applied methodology. Analysis of the IL-6 levels in gingival crevicular fluid (GCF) of patients with CP showed a decrease in disease sites, as compared to non-diseased sites In most studies evaluating DCs in periodontal disease, an increase in these cells can be observed in diseased tissues, as compared to healthy samples, although a decrease in the later processes of the disease can also be seen The present study found an increase in the percentage of sites with PD>4 and CAL >3, and IL-6 levels and immature DCs with higher inflammatory infiltrate. In a previous study by our research group, an increase in immature DCs with inflammatory infiltrate was identified, while mature DCs proved to be positively correlated with mild inflammatory infiltrate in samples presenting chronic gingivitis In conclusion, the IL-6 can contribute to the increase of the immature DCs in the CP, in turn impacting the inflammatory response. Higher levels of IL-2, TNF-α, INF-


N2 nucleophilic substitution reaction involving the replacement of the ester group on the monomer with a secondary amide linkage. On the other hand, GTA could react with free amino groups of chitosan, leading to the formation of either the Schiff bases or the Michael-type adducts with terminal aldehydes. The biocompatibility of non-cross-linked chitosan membranes was demonstrated by the absence of any signs of toxicity or inflammation reaction. The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples. In addition, the materials modified with GTA trigger apoptosis at an early stage and may induce toxicity in the RPE cells later. The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated. In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.The interaction between cells and biopolymers is the evaluation indicator of the biocompatibility of materials. The purpose of this work was to examine the responses of retinal pigment epithelial (RPE) cells to genipin (GP) or glutaraldehyde (GTA) cross-linked chitosan by means of cell viability assays, cytokine expression analyses, and apoptosis assays. Evaluations of non-cross-linked chitosan were conducted simultaneously for comparison. Both GP and GTA treated samples with the same extent of cross-linking (around 80%) were prepared by varying cross-linking time. Our results showed that GP cross-linking was carried out by either radical polymerization of the monomers or S Retinaltoxicity . These sd-glucosamine units and shows antibacterial and wound healing activities [et al. [Chitosan is a naturally occurring cationic polysaccharide obtained by deacetylation of chitin, a biopolymer second in abundance to cellulose. It is primarily comprised of repeating tivities ,8. As a tivities ,10. In ttivities . A recen [et al.  has prov [et al. .i.e., genipin (GP) and glutaraldehyde (GTA) are more feasible for chemical modification. It is known that although cross-linking is a common method to increase the stability of biomaterials, the treatment involving the use of chemical cross-linkers probably causes toxic side-effects [Cross-linking is recommended to overcome the hydrophilic nature of chitosan for its application as a drug carrier . Water-s-effects . Several-effects , human f-effects , L929 mu-effects , Caco-2 -effects , and pig-effects . Howeverin vitro study to examine the responses of retinal pigment epithelial (RPE) cells to GP or GTA treated chitosan. Evaluations of non-cross-linked chitosan samples were conducted simultaneously for comparison. The information from cell-material interactions that is relevant to ocular biocompatibility will facilitate the engineering of the appropriate cross-linked chitosan to enhance the delivery of drug therapeutics.In view of the potential of the development of cross-linked chitosan-based drug delivery systems for the treatment of posterior segment diseases such as AMD, diabetic retinopathy, and cytomegalovirus retinitis, there must be concern about the possible safety issues related to chemical cross-linking of biomaterials. Here, we performed an 2.2.1.N2 nucleophilic substitution reaction involving the replacement of the ester group on the monomer with a secondary amide linkage. The detailed reaction pathways for GP cross-linking of amino group containing biopolymers has been discussed by Butler and colleagues [In this study, to cross-link the chitosan materials, we adopted a film immersion method as previously described for the chemical modification of gelatin sheets  and humalleagues . On the p < 0.05). A similar trend was found for the GTA-chi groups. At each time point, the cross-linking index of chitosan samples treated with GTA was significantly higher than their counterparts with GP (p < 0.05), which indicated that the GTA demonstrates a higher reactivity during chemical cross-linking. These findings are in accordance with previous results showing that the GTA produces stronger cross-linking to gelatin molecules than using GP [i.e., 78.5 ± 2.8% for GP-chi at 8 h and 76.8 ± 2.6% for GTA-chi at 3 h) were characterized by various in vitro assays.The ninhydrin assay has been shown to be applicable for determining the extent of cross-linking in amino group containing biomaterials ,13. In tusing GP . It is kusing GP , which m−1 are shown in −1 (C=O stretching in amide I), 1550 cm−1 (N-H bending in amide II), 1585 cm−1 (N-H bending in non-acetylated 2-aminoglucose primary amine), and 1151 cm−1 (C-O-C asymmetric stretching), which are characteristic of chitosan polysaccharide structure [−1, which were assigned to the C-O-C asymmetric stretching and the CH3 bending of the methyl ester, and C=C ring stretching, respectively. These data are in agreement with previous FTIR study on GP treated biopolymers [−1, indicating the presence of C=N bonds from the Schiff bases. Furthermore, a new peak appeared at 1729 cm−1 which, according to the literature [Before cytocompatibility tests, both above-mentioned samples with the same cross-linking degree were studied by Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, which is a useful tool for the identification of molecular interactions in the chemically modified biomaterials . In thistructure . After Gpolymers . The spe2.2.p > 0.05) after three days in culture. In addition, the viability of these RPE cell lines was greater than 96%. In contrast, the mean percentage of live cells was significantly reduced by about 86% (p < 0.05) for GTA-chi as compared to those of the control groups. The ARPE-19 cells exposed to GTA cross-linked chitosan membranes had relatively higher cytotoxicity than did those to GP treated samples.in vitro and in vivo assays [in vitro, chemical cross-linking may induce cell/tissue responses to biomaterials. As shown in the literature, GTA has been used to modify different biopolymers such as gelatin [et al. [versus GTA treated counterparts. Consistent with these data, we showed that the chitosan materials cross-linked with GP, in contrast to those with GTA, are more compatible toward human RPE cells and have potential suitability for application as drug carriers in the treatment of posterior segment diseases.Chitosan is a well-known biocompatible and bioactive material and has been under investigation for a vast array of biomedical applications including sutures, wound dressings, tissue engineering scaffolds, and drug/gene delivery vehicles . Severalo assays ,33. The  gelatin , hyaluro gelatin , and chi gelatin  for crea gelatin ,16. In t [et al.  have rep2.3.p < 0.05). On the other hand, p < 0.05).IL-6, which appears to be a major cytokine-triggering mediator in the later part of the acute phase, can be used as an indicator of biocompatibility of the materials tested . For evaet al. [et al. [et al. [in vitro ocular biocompatibility of chitosan materials. However, our data also show that the GTA cross-linked chitosan membranes have a marked stimulatory influence on IL-6 production. More recently, we investigated the pro-inflammatory cytokine expression elicited in ARPE-19 cells in response to GTA cross-linked gelatin hydrogels and found similar effects [p > 0.05). It is known that GP is a naturally occurring cross-linker and has been widely applied in herbal medicine and food industry [et al. [Chatelet et al.  have rep [et al.  and Vand [et al.  also dem effects . The treindustry . Koo et  [et al.  have rep2.4.p > 0.05). In the case of Chi and GP-chi groups, only approximately 1% of the cells were positively labeled with TUNEL. These results indicate that the non-cross-linked and GP cross-linked chitosan materials do not cause apoptosis in RPE cells. The proportion of apoptotic cells was significantly higher in cultures treated with GTA cross-linked chitosan membranes compared with those exposed to GP cross-linked samples (p < 0.05), suggesting that cross-linking of biomaterials with GTA may be relevant for apoptosis induction.in vivo animal model was previously established to assess the inflammatory response of the eye to biomaterials [in vitro cellular responses to various chitosan materials using ARPE-19 cultures before further in vivo animal experiments. The results of both cell viability assays and pro-inflammatory gene and cytokine expression analyses indicate that the GP cross-linked chitosan membranes have good compatibility towards human RPE cells. On the contrary, the samples cross-linked with GTA show cytotoxicity and are not suitable for intraocular application. It has been reported that cross-linker toxicity to osteoblasts cultured on GTA treated collagen/poly films is by the mechanism of apoptosis [et al. [Our group recently developed an intraocular cell delivery system using biopolymer-based carriers ,16,37–39aterials . Here, wpoptosis . In acco [et al.  also dem3.3.1.Chitosan (Cat. No. 50494), derived from crab shell, was a commercial powder supplied by Fluka . According to information from the manufacturer, the chitosan samples used as raw materials had a degree of deacetylation of 95–98% and a molecular weight of approximately 150 kDa. Glutaraldehyde and ninhydrin reagent were purchased from Sigma-Aldrich . Genipin was purchased from Wako Pure Chemical Industries . Phosphate-buffered saline  was obtained from Biochrom AG . Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture (DMEM/F12) and TRIzol reagent were purchased from Gibco-BRL . Fetal bovine serum (FBS) and the antibiotic/antimycotic (A/A) solution  were obtained from Biological Industries . All the other chemicals were of reagent grade and used as received without further purification.3.2.The chitosan membranes (Chi group) were prepared by solution casting method. In brief, chitosan (1 g) was added to 1% v/v aqueous acetic acid (50 mL) with stirring until complete dissolution. To remove insoluble substance, the solution was passed through a filter paper . Then, 0.5 mL of chitosan solution was poured in a well of a 24-well plate  and air-dried for 2 days at 25 °C to obtain membranes (about 10 μm thick). The membrane samples were incubated in a 0.5 N NaOH solution for 1 h and washed extensively with deionized water until neutrality.in vacuo for 24 h and sterilized in a graded series of ethanol solutions and thoroughly rinsed in sterilized PBS for use in the in vitro experiments.The chitosan materials were treated with GP (GP-chi group) or GTA (GTA-chi group) by respectively immersing the membrane samples in 5 mL of PBS containing 10 mM cross-linker. The cross-linking reaction was allowed to proceed at 25 °C for different time periods (0–24 h). To eliminate the residual GP or GTA, the cross-linked membranes were washed extensively in deionized water. Then, the samples were dried Cb) and after (Ca) cross-linking is proportional to the optical absorbance of the solution. The extent of cross-linking of the chitosan membranes was calculated as cross-linking index (%) = ((Cb − Ca)/Cb) × 100. Results were averaged on five independent runs.The amount of free amino groups of chitosan membranes was determined to evaluate their extent of cross-linking . The tes−1 with a resolution of 4 cm−1.The FTIR spectroscopy of various chitosan membranes was performed using a FT-730 ATR-FTIR Spectrophotometer  according to the previously published method . The spe3.3.2 at 37 °C. The cells from passage 38 were used for experiments.ARPE-19 cells, a spontaneously immortalized human cell line (BCRC No. 60383) with morphological and functional characteristics similar to adult human RPE , were pu4 cells/well) were seeded in 24-well plates and maintained at 37 °C with 5% CO2. Using cell culture inserts , each well of a 24-well plate was divided into two compartments. A 1-cm2 membrane sample (5 mg) was then placed into the inner well of the double-chamber system to examine the cells cultured on the plastic plate. At specific time intervals, the qualitative and quantitative assays were performed following removal of the inserts and chitosan samples. ARPE-19 cells in regular growth medium without test materials served as control groups.For evaluation of cellular responses to cross-linked chitosan materials, an indirect contact methodology was used as described elsewhere . In brie3.4.ARPE-19 cells were grown to confluence and then were exposed to various chitosan membranes for 3 days. Cell viability was determined using the Live/Dead Viability/Cytotoxicity Kit from Molecular Probes  . This as3.5.15 primers (Promega). The primers used to amplify the human IL-6 complementary DNA (cDNA) were 5′-CCACTCACCTCTTCAGAACGAA-3′ (sense) and 5′-GGCAAGTCTCCTCATTGAATCC-3′ (antisense), and those used to amplify the internal control cDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were 5′-TGGTATCGTGGAAGGACTCATGAC-3′ (sense) and 5′-ATGCCAGTGAGCTTCCCGTTCAGC-3′ (antisense). Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was performed on a Light-Cycler instrument  according to the manufacturer’s instructions with FastStart DNA Master SYBR Green I reagent (Roche Diagnostics). Each sample was determined in triplicate and the results for IL-6 were normalized to the level of GAPDH mRNA. On the other hand, aliquots of the supernatant were collected to measure the IL-6 levels. The release of IL-6 from cultivated cells into the conditioned medium was detected by the Quantikine enzyme-linked immunosorbent assay (ELISA) kit  specific for human IL-6. Cytokine bioassays were performed according to the manufacturer’s instructions. Photometric readings at 450 nm were measured using the Spectrophotometer . Results were expressed as pg/mL. All experiments were conducted in quadruplicate.ARPE-19 cells were grown to confluence and then were exposed to various chitosan membranes for 3 days. Interleukin-6 (IL-6) expression was detected at both messenger RNA (mRNA) and protein levels . Total R3.6.After seeding overnight, the attached RPE cells were exposed to various chitosan membranes for 24 h. Apoptotic cells in ARPE-19 cultures were determined using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay (Roche Diagnostics). Samples were fixed with 4% paraformaldehyde for 1 h at room temperature. After washing with PBS, the fixed specimens were permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice and incubated with a mixture of TdT solution and fluorescein isothiocyanate dUTP solution in a humidified chamber for 1 h at 37 °C. The negative controls were incubated with distilled water in place of TdT enzyme. Cells treated with DNase served as positive controls. To visualize cell nuclei, the samples were stained with 4′,6-diamidino-2-phenylindole  and examined microscopically at 400× magnification. Ten different areas were randomly selected, and the number of TUNEL-positive cell nuclei was quantified. All experiments were conducted in triplicate, and the apoptotic index was calculated as the average ratio of apoptotic cells to the total number of cells in these 30 different areas.3.7.p < 0.05.Results were expressed as mean ± standard deviation. Comparative studies of means were performed using one-way analysis of variance (ANOVA). Significance was accepted with 4.in vitro cellular responses to chemically modified chitosan samples. The results suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated. In terms of material safety, the GP cross-linked chitosan may have biocompatibility for potential application as delivery carrier in the treatment of posterior segment diseases.In this work, cell viability assays, cytokine expression analyses, and apoptosis assays were performed to examine the interaction between human RPE cells and various chitosan materials. The biocompatibility of non-cross-linked chitosan membranes is demonstrated by the absence of any signs of toxicity or inflammation reaction. We have also investigated the effect of cross-linker functionality on the


No differences in the amplitude of the response were observed between NAcshell and NAccore compartments, though the dopaminergic response in the NAcshell was transient whereas that in the NAccore rose slowly to reach a plateau. These results demonstrate the feasibility to use multiprobe voltammetry to measure discrete monoaminergic responses in discrete areas of the brain and confirm the effect of D3 receptors antagonist at modifying the neurochemical effects of cocaine.Dopamine (DA) D3 receptors have been associated with drug intake and abuse and selectively distribute in the brain circuits responding to drug administration. Here we examined the effects of an acute systemic administration of cocaine (15 mg/kg) alone or preceded by treatment with the selective D3 receptor antagonist SB-277011-A (10 mg/kg) on DA levels concurrently in the rat nucleus accumbens shell and core sub-regions . It is shown that cocaine increases extracellular DA in both compartments and that blocking D3 receptors with SB-277011-A, although the latter is devoid of dopaminergic effects  The mesIn particular, the shell establishes intense connections with the limbic system and the hypothalamus and is particularly responsive to the reinforcing properties of addictive compounds, . DiffereIn the early 90s DA metabolism was shown to be increased in the shell but not in core following mild stress. In contrast, antipsychotic drugs such as haloperidol were found to increase DA metabolism more in the core than in the shell, whereas clozapine produced approximately the same effect on DA metabolism in both subregions. Based on such findings, it was concluded that the shell relates to mesolimbic pathway involved largely with emotional and motivational processes, while the core is functionally related to the striatal area and plays a role predominantly in motor functions , 9.et al. [In addition, Cadoni et al.  have demet al. , yet wheet al. . Besideset al. . In contet al. .Altogether, the data mentioned support the indication that the accumbens core is densely linked to the neostriatum in terms of input, structure and contribution in motor function. In contrast, the shell is linked with limbic and subcortical areas, is considered to be a component of the ‘extended amygdala’ and to play a role in motivation and emotional responding .in vivo data cited above have been obtained with the microdialysis methodology. Likewise, very few studies in NAccore and shell have been reported up to date with the other major in vivo electrochemical methodology i.e. voltammetry. For instance, a very short lasting (seconds) increase of the catecholaminergic related voltammetric signal in shell but not in core in rats given free-choice access to a novel environment has been described [In particular, most of the escribed . On the escribed .1.2.The NAc contains high density of the D2-like family of dopamine receptors to which belong the D3 receptors. To a lesser extent, D3 receptors are expressed in the amygdala,  and muchUnder physiologic conditions the extrasynaptically located DA transporter (DAT) affects extrasynaptic DA levels helping terminate DA action and favouring its recycling . Cocainein vitro D3/D2 affinity ratio for human and rat of 120 and 80, respectively and with a 100-fold selectivity over 66 other receptors, enzymes, and ion channels [SB-277011-A is a selective D3 receptor antagonist with high affinity for human (pKi 7.95) and rat (pKi 7.97) cloned DA D3 receptor, with an channels .The compound has a good brain penetration , which acknowledges the European Directive 86/609/EEC, and were fully compliant with GlaxoSmithKline policy on the care and use of laboratory animal and codes of practice.Adult male CD rats (250-310 g) were employed in this study. Rats were subjected to a normal light/dark cycle (light on at 7.00 a.m.) with food and water available 2.2.Following basal recording for 30 min, the animals were randomly assigned to one of the three treatment groups. Animals from group 1 received systemic  administration of vehicle , group 2 animals received vehicle and cocaine  and animals from group 3 received SB-277011A . SB-277011A and cocaine were both dissolved in cavasol 10%.2.3.Voltammetry was applied by means of a μAutolab polarograph  linked to an IBM PC computer equipped with a General-Purpose Electrochemical System Software (GPES) package. Voltammetric measurement was performed with a three-electrode potentiostat system made of a silver/silver chloride (Ag/AgCl) reference electrode, a copper wire auxiliary (counter) electrode both approximately 100 μm in diameter , 31, and2.4.dura mater through two adjacent holes (200 μm diameter) that were drilled in the parietal bone.Rats were anaesthetised with urethane (1.5 g/kg i.p.) and held in a Kopf stereotaxic frame throughout the experiment. Two mCFE were inserted with their active tips in core and shell of the NAc, respectively. The “working” electrodes (mCFE) were placed side by side in a dedicated holder. Their active tips were inserted in each animal ipsilaterally in the cerebral areas studied as calculated from bregma according to Paxinos and Watson : NAccorein vivo based upon in vitro calibration that was performed with Differential Pulse Voltammetry (DPV) associated with Nafion mCFE in solution.The amperometric parameters were selected to achieve real time measurement of DA-like oxidation current -1, scan duration: 6 sec, filter: 0.1 Hz, frequency of scans: every 5 min, filter: 0.1 Hz. The polarograph automatically measured the current (nA) coming from peak oxidation of DA by measuring the perpendicular height (h) from the top of the peak to the tangent line drawn between its shoulders as shown in in vivo amperometric analysis of DA was performed at the potential +90 mV: i.e. the voltage of the potentiostat was increased instantaneously to the selected potential that corresponds to the potential of a complete oxidation of DA in our amperometric conditions. DA-related currents in core and shell (in nanoAmperes) were detected in time intervals of 0.2 Sec.DPV parameters used were as follows: initial potential (Ei) -100 mV, final potential (Ef) +200 mV, scan step was 50 mV.sec2.5At the end of each experiment, an electrolytic lesion  was made through the carbon fibre electrode for histological verification of the recording site. Then the animal was sacrificed, its brain removed and quickly frozen. Each brain was then sliced on a microtome (50 μm), stained (Nissl), and examined under microscope for electrode placement see .2.6.post-hoc test was applied on treatment by time interaction since the factor area was not significant per se nor in interaction with all the other factors. Statistical significance was set at p < 0.05 for all tests.Each 0.2 sec DA measurement was added and averaged over 5 min bins. Averaged data were transformed in percent of the averaged last 15-min basal levels prior to any treatment. Data have been log transformed and statistically analyzed using a three-way ANOVA with between-subjects factors of treatment and NAc sub-region and repeated measurement factor of time using Statistica, version 8.0. Planned comparison 3.Mesolimbic dopamine (DA) neurotransmission plays a central role in the expression of drug of abuse-seeking behaviour, pharmacological effects of drug intake and relapse to repeated drug taking . = 2.6, p = 0.003 and treatment by time interaction F = 4.43, p = 0.001 in the NAccore while in the NAcshell it revealed a significant effect of time F = 2.12, p = 0.018 and treatment by time interaction F = 3.03, p = 0.001. However, no significant difference was observed between shell and core when comparing the effect of cocaine on DA release in both areas. Thus the data from each animal were meaned across regions and values were plotted for comparison versus vehicle and versus SB-277011-A treated rats.No statistical differences were observed between these areas following two successive vehicle i.p. treatments , performed at 30 and 120 min of recording period, respectively, thus vehicle data have been plotted to perform the comparison vehicle versus drug treatments. Similarly, treatment with SB-277011-A  did not alter significantly DA levels in both brain areas studied see  top. In  = 5.14, p = 0.013, time F = 5.65, p = 0.001 and treatment by time interaction F = 10,62, p = 0.001  and SB-277011-A + cocaine (group 3) at times 80 — 95 min.In group 3, i.e. in rats that received pre-treatment with the D3 antagonist SB-277011-A (10 mg/kg i.p.) followed by acute cocaine administration (15 mg/kg i.p.) showed larger increase in both areas of the DA levels than that monitored after cocaine alone, as shown in  = 0.001 . Finally3.1.1.in vivo microdialysis results have reported either no significant differences in core and shell extracellular DA levels [et al. [A number of reports indicate differences in shell and core DA metabolism , 9, DA rA levels  - 48, orA levels , 50 or, A levels . ConsequA levels . In thisA levels . In this [et al. , as well [et al. .However, another voltammetric study proposed considerably higher concentration of basal DA levels in NAccore, i.e. in the micromolar range . The dif3.1.2.The present work also shows that acute treatment with 15 mg/kg ip cocaine resulted in a similar increase in extracellular DA levels in both core and shell. Again, this result is in accord with the above mentioned meta -analysis study  indicatiin vivo findings confirm that the electrochemical signal measured in this study is due to the oxidation of DA and not of DOPAC.Furthermore, these data confirm the effective selectiveness of the Nafion biosensors used as it is known that while cocaine is increasing DA levels in brain areas, it also produced a significant decrease in dihydroxyphenylacetic acid (DOPAC) levels , 56. ThePrevious works have shown that DA concentrations were high in the nAc, and that noradrenaline (NA) and serotonin (5-HT) concentrations were considerably lower than the DA concentration . In part3.1.3., voltammetric DA levels measured in both core and shell subregions were not significantly affected by treatment with SB-277011-A. However, pre-treatment with SB-277011-A induced a significant boost of the effect of a subsequent systemic cocaine administration in respect to the effects of cocaine alone  in the NAc was enhanced by SB-277011-A pre-treatment while the compound alone produces only limited changes in rCBV [The dopamine D3 receptor is mainly expressed in regions of the brain associated with the limbic system and in particular in the shell of the NAc -61. Thesne alone . This in in rCBV .Although PK direct interaction between cocaine and SB-277011-A can not be excluded the augmented hemodynamic response reported  is consiet al. [Altogether, these data suggest that the dopamine D3 receptor might mediate an inhibitory action on downstream activity in limbic circuits and that blockade of such receptor by SB-277011-A may enhance the effect of cocaine i.e. via increased extracellular DA concentration in a number of brain regions as reported by Schwarz et al. . This maet al. ) or/and All these observations concur with the present evaluation of a modulating influence of D3 receptor antagonism on the cocaine effect on DA activity in the nAc.4.This work demonstrates that this new dual-probe mCFE approach can be used to monitor extracellular levels of DA in discrete brain regions as reduced in dimension and interconnected as core and shell of NAc. Further, this technique can discriminate between discrete neurochemical changes occurring in response to pharmacological challenge(s).Main evidence of the present work is that acute treatment with 15 mg/kg i.p. cocaine resulted in a similar increase of extracellular DA levels in both core and shell. This data is in accord with the most recent meta-analysis on in vivo microdialysis studies on cocaine–induced DA overflow in the NAc . AnotherThis interpretation agrees with the recent suggestion that the dopamine D3 receptors are more implicated in the motivation to self-administer drugs of abuse than in their direct reinforcing effects  and withFinally, these data, together with the reported efficacy of such a compound on attenuating reinstatement of drug-seeking behaviour  leads on


Generally, bone formation increased with increasing peak force. At the endocortical surface, contrary to our hypothesis, aged mice had a significantly greater response to loading than young-adult mice . Responses at the periosteal surface did not differ between age groups (p > .05). The loading-induced increase in bone formation resulted in increased cortical area in both age groups . In contrast to the strong cortical response, loading only weakly stimulated trabecular bone formation. Serial (in vivo) µCT examinations at the proximal metaphysis revealed that loading caused a loss of trabecular bone in 7-month-old mice, whereas it appeared to prevent bone loss in 22-month-old mice. In summary, 1 week of daily tibial compression stimulated a robust endocortical and periosteal bone-formation response at the mid-diaphysis in both young-adult and aged male BALB/c mice. We conclude that aging does not limit the short-term anabolic response of cortical bone to mechanical stimulation in our animal model. © 2010 American Society for Bone and Mineral Research.With aging, the skeleton may lose its ability to respond to positive mechanical stimuli. We hypothesized that aged mice are less responsive to loading than young-adult mice. We subjected aged (22 months) and young-adult (7 months) BALB/c male mice to daily bouts of axial tibial compression for 1 week and evaluated cortical and trabecular responses using micro–computed tomography (µCT) and dynamic histomorphometry. The right legs of 95 mice were loaded for 60 rest-inserted cycles per day to 8, 10, or 12 N peak force . At the mid-diaphysis, mice from both age groups showed a strong anabolic response on the endocortex (Ec) and periosteum (Ps) [Ec.MS/BS and Ps.MS/BS: loaded (right) versus control (left),  Rates of bone formation diminish with aging in humans and mice.–4 One rePrior studies of direct skeletal overloading in aged animals used approaches that were either invasive  or loadThe right tibias of adult male BALB/c mice  were subjected to noninvasive axial compression based on a method described by others.,17 Mice n = 5) and 22 (n = 4) months using a combination of strain-gauge measurements, µCT imaging, and engineering beam theory. Our goal was to determine force values that produced peak endocortical strains in the 800- to 2000-µɛ range. Owing to the curvature of the mouse tibia, axial compression generates combined compression-bending at the mid-diaphysis. Previous analysis indicated that the site of peak axial strain is near the mid-diaphysis, approximately 5 mm proximal to the distal tibiofibular junction (TFJ).(r2 > 0.85). Tibias then were scanned at the gauge site by µCT . From cross-sectional images, the bone centroid, area, and moment of inertia about the neutral axis were determined, as well as distances from the centroid to the gauge locations (ygauge) and to the predicted sites of peak endocortical and periosteal strain (y*) (gauge(y*/ygauge), where * denotes the site of interest . Compressive and tensile values were based on the gauges on the compressive and tensile sides, respectively. From this analysis, we selected force levels of 8, 10, and 12 N for in vivo loading , and thus values of strain for a given force were similar for 7- and 22-month tibias (p > .05).Prior to in vivo loading, we determined force-strain relationships for tibial compression in mice at ages 7 (on (TFJ). In the ton (TFJ).A. Two siain (y*) B. Based  loading ; Fig. 1Cn = 36; 22 months: n = 35). Under anesthesia, the right legs were loaded to a peak force of 8, 10, or 12 N for 60 cycles/day for 5 days (M–F); left legs were not loaded (control). To label new bone formation, mice were injected with fluorochromes on days 4  and 9  and killed on day 11 by CO2 asphyxiation. Seven mice died prior to the end of the study: Two died from anesthesia overdose, one was euthanized to relieve distress, and four were euthanized after sustaining a tibial fracture during loading. For the remaining 64 mice, bilateral tibias were harvested immediately postmortem, fixed for 24 hours in 4% paraformaldehyde, and stored in 70% ethanol.We performed in vivo loading on 71 mice (7 months: p < .001) and at the end of the experiment . Mice lost an average of 4% (7 months) and 7% (22 months) of body weight during the experiment. Importantly, body weights did not differ between force groups (p > .05) either at the start or the end of the study.Body weights differed slightly between age groups at the start of the study . The volume of interest was located just distal to the physis, spanning a height of 480 µm (30 slices) and containing all bone inside the cortical shell. Using the manufacturer's 3D analysis tools, we determined apparent volumetric bone mineral density (vBMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) . A threSingle-labeled surface was defined as surface labeled with red, green, or yellow (red and green overlaid without separation). Double-labeled surface was defined where both red and green labels were present with separation. MS/BS was calculated as 0.5 × sLS/BS + dLS/BS for all samples. If a sample had no double-labeled surface (dLS/BS = 0), it was coded as “no data” for MAR and BFR/BS based on a published recommendation.. For analendation. We also n = 12) were loaded to a peak force of 12 N on days 1 through 5. Mice aged 22 months (n = 12) were loaded to a peak force of 12 N on day 1 and 10 N on days 2 through 5; the force level was reduced because three 22-month-old mice sustained fractures on day 1. Another two 22-month-old mice died during the study from anesthesia overdose. After the second µCT scan, mice were killed, and bilateral tibias harvested. Tibias were decalcified and embedded in paraffin. Midsagittal sections (5 µm) were cut and stained for tartrate resistant acid phosphatase (TRACP) 3 days prior to loading. Tibias were loaded for 5 days (M–F) using the same protocol as study 1 and rescanned 4 days after the end of loading (the time of the second fluorochrome label in study 1).  Because results of study 1 (see below) showed no effect of force level on trabecular parameters, we used a single force level for study 2. Mice aged 7 months (e (TRACP) to labelt tests . Effects of age and force level were assessed by two-way analysis of variance (ANOVA). ANOVA was performed on absolute data from the left and right legs as well as on relative (r) data . When the overall ANOVA reached significance for force level, post hoc differences were evaluated using Fisher's protected least squares difference (PLSD) test. For study 2, changes with time were assessed using paired t tests (before versus after). Differences were defined as statically significant at p < .05.Differences between loaded (R) and nonloaded (L) legs were assessed by paired p < .001), whereas cortical area was 18% less (p < .001), and cortical width was 23% less (p < .001). Trabecular BV/TV was 54% less in 22- versus 7-month-old tibias; Tb.N, Tb.Th, and vBMD were 6%, 10%, and 34% less . Periosteal indices did not differ between age groups.Analysis of cortical bone formation in nonloaded tibias revealed single-labeled surface in all bones, but a low incidence of double-labeled surface in both age groups. Only 5 of 59 bones had endocortical double label , and onlendocortical surface, in both 22- and 7-month-old mice, loaded tibias had greater Ec.dLS/BS and Ec.MS/BS values than control tibias . ANOVA of relative differences (loaded to control) also indicated that r.Ec.dLS/BS and r.Ec.MS/BS values were greater in 22- than in 7-month-old mice (p < .001).  On the periosteal surface, there also was a significant loading effect in both 22- and 7-month-old mice, with Ps.sLS/BS, Ps.dLS/BS, and Ps.MS/BS values significantly greater in loaded versus control tibias  in loaded tibias revealed that contrary to our hypothesis, aged mice did not have diminished responsiveness to loading  and 3. Bp < .01; , indicatendocortical surface of loaded tibias, Ec.dLS/BS and Ec.MS/BS values depended on force level (p < .05 by two-way ANOVA), whereas on the periosteal surface, Ps.sLS/BS, Ps.MS/BS, Ps.MAR, and Ps.BFR/BS values depended on force level (p < .05). For example, for 7-month-old mice, loaded tibias in the 10- and 12-N groups had significantly greater Ps.sLS/BS and Ps.BFR/BS values than those in the 8-N group; for 22-month-old mice, loaded tibias in the 12-N group had significantly greater Ps.dLS/BS and Ps.MS/BS values than those in the 8-N group .The increase in bone-formation rates induced by mechanical loading resulted in increased cortical area and width in both age groups. In 7-month-old mice, cortical area was 5% greater in loaded tibias than in nonloaded controls . There were no significant effects of force level on trabecular µCT parameters for either age group. Thus loading appears to have caused loss of trabecular bone in 7-month-old mice but a marginal increase in 22-month-old mice.Trabecular bone formation in the proximal tibial metaphysis was modestly enhanced by loading, whereas trabecular structure was strongly negatively affected. In 7-month-old mice, loaded tibias in the 10- and 12-N groups had a significantly higher trabecular MS/BS compared with controls . Similarp = .048) but not in control tibias (p = .82), indicating that loading caused loss of trabecular bone (p = .051) and vBMD (p = .09). In contrast, in 22-month-old mice, trabecular BV/TV did not change with time in loaded tibias (p = .88) but decreased significantly with time in control tibias (p = .002), suggesting that loading prevented bone loss in this age group. Finally, histomorphometric analysis of TRACP-labeled osteoclasts did not reveal differences in osteoclast surface between loaded and control tibias  and young-adult (7 months) mice to daily bouts of axial tibial compression for 1 week and evaluated their cortical and trabecular responses. We hypothesized that aged mice would be less responsive to loading than young-adult mice. At the tibial diaphysis, mice from both age groups showed a strong anabolic response, with a general finding of increasing bone formation with increasing load magnitude. Contrary to our hypothesis, at the endocortical surface, aged mice had a significantly greater response to loading than young-adult mice. Responses at the periosteal surface did not differ between age groups. At the proximal metaphysis, loading caused a loss of trabecular bone in young-adult mice, whereas it appeared to prevent bone loss in aged mice. Taken together, our results indicate no loss of short-term mechanoresponsiveness from age 7 to 22 months in the tibias of male BALB/c mice.Our findings are in contrast to results of three previous studies that also used direct loading but concluded that cortical bone in the aged skeleton is relatively unresponsive. Rubin and colleagues compared the responses of aged (3 years) versus young-adult (1 years) turkeys to axial compression of the surgically isolated ulna  and concluded that a loading stimulus “that is clearly osteogenic in the young-adult skeleton is hardly acknowledged in older bone tissue.” SimilarlThe reasons for the difference in our findings from those of prior studies are unclear. We do not believe that it can be attributed to loading history. The range of strain magnitudes we applied  is comparable with that used by Turner and colleagues. At the lAlthough our findings are at odds with previous direct loading studies, they are consistent with several exercise studies. Two studies found that aged rats were more responsive to weight-bearing exercise than young-adult rats.,11 In onWhereas the cortical response to tibial compression in our study was strongly anabolic, the trabecular response was not. Dynamic histomorphometry indicated that loading modestly enhanced some measures of trabecular bone formation, yet µCT revealed a loss of bone in 7-month-old mice and no gain in 22-month-old mice. Taken together, these results suggest an increase in trabecular bone resorption in the 7-month-old group, although we were unable to detect an increase in osteoclast surface. Our trabecular findings are at odds with some, but not all, previous reports using the tibial compression model. Two-month-old mice had increased bone in the tibial metaphysis after 2 or 6 weeks of loading.,32 and iThe trabecular results from our study and other studies using the tibial compression-loading model should be considered with the caveat that the loading environment in the trabecular bone is unknown. Whereas strains at the cortical mid-diaphysis can be measured with gauges, and simple equations then can be used to estimate peak endocortical and periosteal strains, the same approach cannot be applied to the metaphysis. Even if strain gauges were applied to the metaphyseal surface, they would not provide a measure of the local strains in the endosteal compartment. Finite-element analysis could be used to estimate trabecular strains, although this has not yet been reported. Thus, while cortical results between groups and between studies can be compared in the context of applied strain, the same does not hold for trabecular results. For this reason, our primary focus has been on the cortical response, and it is the cortical results that were used to test our hypothesis.We chose 7 and 22 months as examples of young-adult and aged mice. Mice have an average lifespan on the order of 24 months. Their period of rapid skeletal growth ends by 4 months of age, and cortical bone mass is maintained through 12 months.,18,34,35One strength of our study was the use of in vivo µCT to assess temporal changes in trabecular bone. Typically, responses in rodent models of unilateral loading are determined by comparison of loaded versus control tibias, as we did in study 1. The loaded versus control comparisons of study 1 indicated less trabecular bone in loaded tibias (7-month-old group). This is consistent with the temporal decrease in trabecular BV/TV in loaded tibias (together with no change in control tibias) that we observed in study 2. Thus, in the 7-month-old groups the use of serial data was informative but did not lead to a different conclusion. In contrast, the use of serial scanning in 22-month-old mice provided insight not possible from postmortem scans. Trabecular BV/TV was not different between loaded and control tibias at the end of loading (studies 1 and 2). Yet serial scans revealed that there was a decrease in BV/TV in control but not in loaded tibias from 22-month-old mice, leading to a conclusion that loading appeared to prevent bone loss in this age group.Several limitations should be considered when interpreting our findings. First, the strain magnitude produced by a given force tended to be less in the 22-month-old group than in the 7-month-old group. This is consistent with the trend for a greater moment of inertia in the older mice. However, because the strains values were not significantly different between groups, we chose not to adjust the force levels between groups. Had we increased the force level on the aged mice to produce a higher strain, it is likely we would have enhanced their bone-formation response, lending even further support to the conclusion that aged mice responded to loading. Second, we did not perform individualized strain analysis on each mouse that was subjected to in vivo loading. Thus the actual strains generated in these animals may have differed slightly from the estimates based on a priori analysis of a different set of mice. This may have contributed to variability in our results. Third, the force levels applied to the 7- and 22-month-old groups in study 2 were different. Because the trabecular results from study 1 were not affected by force level, it is unlikely that the difference in force level in study 2 had a significant effect. Lastly, we examined a 5-day loading period, which is relatively short compared with other studies of direct loading.–14,16,17In summary, 1 week of daily tibial compression stimulated a robust endocortical and periosteal bone-formation response at the mid-diaphysis in both aged and young-adult male BALB/c mice. At the proximal metaphysis, loading appeared to maintain trabecular bone in aged mice while causing loss of trabecular bone in young adults. We conclude that aging does not limit the short-term anabolic response of cortical bone to mechanical stimulation in our animal model.


Bevacizumab, a 149-kDa protein, is a recombinant humanized monoclonal antibody to VEGF. PEDF, a 50-kDa glycoprotein, has demonstrated anti-vasopermeability properties. In this study, we demonstrated that the combination of bevacizumab and plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18 (p-PEDF-SAINT-18) has a favorable antiangiogenic effect on corneal NV. Four groups  of bevacizumab + p-PEDF-SAINT-18 were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus on the temporal side. Then, 1 μg of p-bFGF-SAINT-18 was prepared and implanted into the rat corneal stroma 1.5 mm from the limbus on the same side. The inhibition of NV was observed and quantified from days 1 to 60. Biomicroscopic examination, western blot analysis and immunohistochemistry were used to analyze the 18-kDa bFGF, 50-kDa PEDF and VEGF protein expression. No inhibition activity for normal limbal vessels was noted. Subconjunctival injection with the combination of bevacizumab and p-PEDF-SAINT-18 successfully inhibited corneal NV. The bFGF and PEDF genes were successfully expressed as shown by western blot analysis, and a mild immune response to HLA-DR was shown by immunohistochemistry. We concluded that the combination of bevacizumab and p-PEDF-SAINT-18 may have more potent and prolonged antiangiogenic effects, making it possible to reduce the frequency of subconjunctival bevacizumab administration combined with a relatively safe profile and low toxicity. NV or angiogenesis, the formation of new blood vessels, is a normal process that accompanies tissue growth, reproduction and the repair of damaged tissue during the process of wound healing. Corneal NV can occur physiologically or pathologically and usually leads to vision impairment ,2. SeverVEGF is a critical mediator of NV, and the level of VEGF in the vitreous is highly correlated with the growth of new blood vessels in eyes with diabetic retinopathy and choroidal NV –11. AntiBevacizumab  is a humanized monoclonal antibody to VEGF that is approved by the U.S. Food and Drug Administration for the treatment of metastatic colorectal cancer . This dret al.methylpyridinium-chloride) delivery system  was based on a cationic pyridinium head group, showing excellent bio-compatibility. The molecular structure of SAINT-18 was purchased from Synvolux Therapeutics B.V. Each vial was “ready to use” and contained 2 mL of SAINT-18 in water at a concentration of 0.75 mM. Before use, the SAINT-18 was vortexed thoroughly to minimize micelle-size, thereby increasing its complexing efficacy. The relative amounts of DNA to carrier for SAINT-18/DNA (1 μL of SAINT-18 per 1 μg of DNA phosphate) were allowed to form at room temperature. To minimize the loss experienced with liquid reagents during transfection into corneal tissue, the complex was partially dried by SpeedVac  at ambient temperature for 60–90 min after the plasmid bFGF (1 μg) and PEDF  had been complexed with SAINT-18. The partially dried form of the complex was drawn out of the Eppendorf tube by curettage and prepared for immediate implantation into the corneal pocket.The SAINT-18 (1-methyl-4-[2/room air mixture (1:1). As additional topical anesthesia, 0.4% benoxinate hydrochloride  was applied to the corneal surface. The eyes were proptosed by grasping the temporal limboconjunctival epithelium with a jeweler’s forceps, and a 30°–45° fan-shaped central-peripheral corneal intrastromal lamellar pocket  was dissected with a surgical blade  and an ophthalmic slit knife . The pocket was extended 1.5 mm from the limbus. After the p-bFGF-SAINT-18 gene complex (1 μg) was implanted into the corneal stromal pocket in each eye using forceps and a blade, 0 + 0 μg, 0.1 + 0.1 μg, 1 + 1 μg and 10 + 10 μg of bevacizumab and p-PEDF-SAINT-18, respectively, were immediately delivered to the subconjunctival tissue 1.5 mm from the limbus via the indwelling cannula using a Hamilton syringe. The topical antibiotic ointment  was applied to the corneal surface to reduce irritation and prevent infection.All surgical procedures were performed using sterile techniques. The corneal pocket implantation performed in this study was a modification of a previously described technique . The ratExaminations were performed with a dissecting microscope, and the results were photographed. While the rats were under anesthesia, the eyes were proptosed, and the maximum vessel length and width in the NV region were measured with calipers. Photographs obtained during corneal angiogenesis assay were taken at a resolution of 640 × 480 pixels using a digital CoolPix 995 camera . The operator was masked to the treatment group of each cornea. The areas containing blood vessels were traced on the computer monitor . The area within the trace was calculated with image analysis software  and is reported in square millimeters with the determinations confirmed at ×40 magnification. Three independent observers conducted the masked assessments.g for 30 min at 4 °C. The protein content of the supernatant was determined using the Bio-Rad Protein Assay system. An equal volume of sample buffer (2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.1% bromophenol blue, 2% 2-mercaptoethanol, 50 mM Tris-HCl (pH 7.2)) was added to the sample. The proteins were separated by electrophoresis  on 15% SDS-polyacrylamide gels at 120 V for 90 min. They were then transferred to polyvinylidene difluoride membrane  in transfer buffer  at 50 V for 60 min. The membrane was blocked with 5% nonfat dry milk in Tween-20 and Tris-buffered saline  for 60 min at room temperature. The membrane was then incubated with anti-bFGF , anti-PEDF antibody  and anti-VEGF  for 90 min at room temperature. Each blot was washed three times for 10 min in TTBS, blocked with 5% nonfat dry milk in TTBS, and then incubated with horseradish-peroxidase-conjugated secondary antibody  for 1 h at room temperature. The antibody labeling was detected by chemiluminescence . Colored molecular-weight standards were run in parallel on each gel. The housekeeping gene α-tubulin was used as the control.On days 6 and 60 after transfection, the rats in each group were killed with an overdose of thiopental sodium, and the fresh corneas were removed along the contour of NV using scissors. These specimens were homogenized by sonication in ice-cold lysis buffer , 150 mM NaCl, 2% Triton X-100, 100 μg/mL phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin) and then centrifuged at 50,000× l-lysine, they were immunostained  with anti-rat HLA-DR antibody .On days 6 and 60 after transfection in group D, the rats were killed with an overdose of thiopental sodium; their eyes were then enucleated, fixed in paraformaldehyde, and frozen at −70 °C in OCT compound. The corneas were excised and cryostatically cut into 3–8-μm sections for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). The excised corneal tissues for IHC were fixed in Bouin’s solution and embedded in paraffin. After the different sections had been cut (3 μm) and mounted on slides coated with poly-p ≤ 0.05 was considered statistically significant.Repeated measures ANOVA  and the Bonferroni post hoc test were used to analyze the differences in the length and area of the corneal NV between pairs of groups. The length and area were measured every three days from day 0 to day 60. The values for each measurement were recorded as separate variables and defined as within-subject factors. The assigned groups were defined as between-subject factors. A value of In conclusion, we found that the combination of bevacizumab and p-PEDF-SAINT-18 may have more potent and prolonged antiangiogenic effects, making it possible to reduce the frequency of subconjunctival bevacizumab administration with a relatively safe profile and low toxicity.